Project Summary DNA viruses such as tailed bacteriophages and herpesviruses infect living cells by delivering their genetic material into the host. This important biological function is mediated by large (M.W. ~ 0.5 - 0.9MDa) oligomeric proteins known as portal proteins. Although different viruses have developed distinct mechanisms for DNA delivery based on host and tissue specificity, portal proteins are well-conserved macromolecules, both in the overall quaternary structure (a ring of 12 identical subunits) and location within the viral capsid, where they replace one of the twelve 5-fold vertices. The long-term-goal of our research is to elucidate the mechanisms of viral DNA delivery into living cells. The short-term goal of our work is to understand the architecture and composition of a prototypical viral DNApumping portal protein and to elucidate the physical and functional interaction of this protein with other virusencoding factors required for DNA injection. To achieve this ambitious task, we will study bacteriophage P22 DNA-delivery machinery. Our research is organized in three specific aims: In Aim 1, we will determine the crystallographic structure of the 12-fold symmetric portal protein of bacteriophage P22 in the conformation adopted in the procapsid (PPPr), as well as in the mature phage (PPMP). This structural work will shed light on the conformational plasticity of the portal protein and define how this molecular machine senses virus maturation by undergoing large conformational switch. In Aim 2, we will characterize the Injection proteins gp7, gp16, and gp20 of bacteriophage P22, which are highly conserved and essential for virus infectivity. Our work will shed light on the chemistry, composition, and assembly of these proteins inside the virion, as well as on the cross talk of these factors with the portal protein. In parallel, we will test the hypothesis that the Injection proteins form a DNA-translocon in the host cell envelope that allows for smooth passage of viral DNA into the host. In Aim 3, we will set up a defined in vitro packaging assay to measure the ATPase activity related to portal protein-mediated DNA pumping. This assay, which does not measure DNA injection per se, but its mirror process, DNA packaging, will allow testing the effect of specific mutations in portal protein, and thus help understand the detailed enzymology of DNA pumping.